The present invention is directed to methods comprising the use of cellulases in industrial processes and compositions therefor. In particular, the present invention is related to treating textiles, e.g., laundering and processing, with cellulase derived from Thermomonospora fusca which is particularly well suited for that purpose. The present invention is further related to the use of cellulase derived from Thermomonospora fusca to enhance the digestibility of animal feed, in detergents, in the treatment of pulp and paper and in the production of starch and treatment of by-products thereof.
Cellulases are enzymes which hydrolyze cellulose (xcex2-1,4-D-glucan linkages) and produce as primary products glucose, cellobiose and cellooligosaccharides. Cellulases are produced by a number of microorganisms and comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (CBH), endoglucanases (EG) and xcex2-glucosidases (BG) (M. Schulein, Methods in Enzymology, vol. 160, pp. 235-242 (1988)). Current theory holds that the enzymes within these classifications can be separated into individual components. For example, microbial cellulase compositions may consist of one or more CBH components, one or more EG components and possibly xcex2-glucosidase. The complete cellulase system comprising CBH, EG and BG components synergistically act to convert crystalline cellulose to glucose. The exo-cellobiohydrolases and the endoglucanases act together to hydrolyze cellulose to small cello-oligosaccharides. The oligosaccharides (mainly cellobioses) are subsequently hydrolyzed to glucose by a major xcex2-glucosidase.
Cellulases and components thereof, used either singularly or in combination, are known to be useful in detergent compositions and for treating textiles. In the textile industry, during or shortly after the manufacture of cotton-containing fabrics, it is known to treat such fabrics with cellulase to impart desirable properties to the fabric. One purpose of this treatment is to remove fuzz, i.e., untangled fiber ends that protrude from the surface of a yarn or fabric, and pills, i.e., bunches or balls of tangled fibers that are held to the surface of a fabric by one or more fibers. Accordingly, in the textile industry, cellulase has been used to improve the feel and/or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, and also for imparting a stone washed appearance to cotton-containing denims. In particular, Japanese Patent Application Nos. 58-36217 and 58-54032 as well as Ohishi et al., xe2x80x9cReformation of Cotton Fabric by Cellulasexe2x80x9d and xe2x80x9cWhat""s Newxe2x80x94Weight Loss Treatment to Soften the Touch of Cotton Fabricxe2x80x9d Japan Textile News, (December 1988) each disclose that treatment of cotton-containing fabrics with cellulase results in an improved feel for the fabric. It is generally believed that this cellulase treatment removes cotton fuzzing and/or surface fibers which reduces the weight of the fabric. The combination of these effects imparts improved feel to the fabric.
Clothing made from cellulose fabric, such as cotton denim, is stiff in texture due to the presence of sizing compositions used to ease manufacturing, handling and assembling of clothing items and typically has a fresh dark dyed appearance. One desirable characteristic of indigo-dyed denim cloth is the alteration of dyed threads with white threads, which gives denim a white on blue appearance. For example, after a period of extended wear and laundering, the clothing items, particularly denim, can develop in the panels and seams localized areas of variation in the form of a lightening in the depth or density of color. In addition, a general fading of the clothes, some pucker in seams and some wrinkling in the fabric panels can often appear. In recent years such a distressed or xe2x80x9cstonewashedxe2x80x9d look, particularly in denim clothing, has become very desirable to a substantial proportion of the public.
Previous methods for producing the distressed look included stonewashing of a clothing item or items in a large tub with pumice stones having a particle size of about 1 to 10 inches and with smaller pumice particles generated by the abrasive nature of the process. Typically the clothing item is tumbled with the pumice while wet for a sufficient period such that the pumice abrades the fabric to produce in the fabric panels localized abraded areas of lighter color and similar lightened areas in the seams. Additionally, the pumice softens the fabric and produces a fuzzy surface similar to that produced by the extended wear and laundering of the fabric.
The use of the pumice stones has several disadvantages, including overload damage to the machine motors, mechanical damage to transport mechanisms and washing drums, environmental waste problems from the grit produced and high labor costs associated with the manual removal of the stones from the pockets of the garments. In view of the problems associated with pumice stones in stonewashing, cellulase solutions are used as a replacement for the pumice stones under agitating and cascading conditions, i.e., in a rotary drum washing machine, to impart a xe2x80x9cstonewashedxe2x80x9d appearance to the denim (U.S. Pat. No. 4,832,864).
A cellulase system derived from the thermophilic, filamentous, soil bacterium Thermomonospora fusca has been detected and the biochemical characteristics of that system and components thereof studied (Wilson, Critical Reviews in Biotechnology, Vol. 12xc2xd, pp. 45-63 (1992)). One specific endoglucanase component of the T. fusca system, E5, has been sequenced (Lao et al., J. Bacter., vol. 173, pp. 3397-3407 (1991)), and its disulfide arrangement and functional domains described (McGinnis et al., Biochemistry, vol. 32, pp. 8157-8161 (1993)). McGinnis discloses that E5 treated with protease from Streptomyces lividans results in a 14 kD cellulose binding domain and a catalytically active 32 kD fragment which had lost the ability to bind to cellulose. Pure catalytically active S. lividans protease treated E5 was shown to have similar activity to intact enzyme on CMC. However, mixtures of catalytically active E5 fragments, when combined with intact E3 from T. fusca or intact E3 and CBHI from Trichoderma reesei, showed decreased performance to similar mixtures containing intact E5 instead of the fragment (PCT Publication No. 96/00281).
Despite intensive research related to the use of cellulases in industrial processes, cellulases known and used in the art have shown significant drawbacks. For example, many cellulases have been problematic due to low activity, poor alkaline or acid stability, poor temperature stability and poor oxidative stability. Surprisingly, Applicants herein have discovered that the E5 cellulase possesses a complement of characteristics which makes its use particularly desirable in certain industrial applications.
According to the present invention, a method of treating cellulosic material is provided comprising contacting the cellulosic material with a cellulase obtainable from Thermomonospora fusca corresponding to E5, a truncated E5, or a derivative thereof. In a process embodiment of the invention, the cellulosic material comprises cellulose containing fabric and the result of the method is to produce a stonewashed effect or an improvement in the feel and/or appearance of the fabric. In an alternative process embodiment of the invention, the cellulose containing fabric is contacted with an aqueous solution containing a detergent composition comprising a cellulase obtainable from T. fusca corresponding to E5, a truncated E5, or a derivative thereof. In yet another process embodiment of the invention, the cellulosic material comprises wood pulp and the addition of cellulase facilitates the production of paper products therefrom. In yet another process embodiment of the invention, the cellulosic material comprises animal feed and the method results in an increase in the digestibility or value of said animal feed. In still further embodiments of the invention, the cellulosic material comprises grain or grain byproducts used in the production of food, starch, ethanol or sugar.
Applicants identify herein a specific cellulase obtainable from T. fusca, known in the literature as E5, having a surprising array of characteristics which are especially beneficial in textile processing (specifically including denim stonewashing and bio-polishing), cleaning products and detergents, pulp and paper production, food processing and as an additive for animal feed. Specifically, Applicants have discovered that E5 has an especially broad pH/activity profile on insoluble substrate, being active in the pH range from about pH 5.0 to 10.5 with very little drop off in activity at the alkaline region. Moreover, E5 has significant activity levels at moderate pH and temperature, is stable for extended periods of time and at temperatures in excess of 80xc2x0 C., is essentially insensitive to many buffer compositions and strengths, remains active after prolonged proteolytic cleavage, and is stable in the presence of oxidants such as perborate and perborate/TAED combinations and in detergents. Among the especially surprising features of E5 are its exceptional high pH activity on insoluble substrate such as cotton fabric compared to its activity on soluble substrate, which decreases significantly after its pH optimum of about 6.0. Thus, E5 is particularly well suited for high pH textile applications, such as simultaneous bleaching and bio-polishing or in laundry detergents and pre- or post-treatments which are formulated for a high pH. Another surprising feature is the ability of T. fusca to remain nearly unaffected by incubation in liquid detergent. An additional novel feature of the present invention is that truncated E5 also possesses many of the same exceptional characteristics as E5. For example, Applicants discovered that a truncated E5 enzyme can exhibit nearly identical surface fiber removal activity as E5.
The considerable advantages of the E5 or truncated E5 cellulase in industrial applications would not have been suggested by the prior art. The invention itself, together with further objects and attendant advantages, will best be understood by reference to the following detailed description taken in conjunction with the accompanying drawings.